The invention relates to methods for measuring the mobility of mass-selected ions in gases at pressures of a few hectopascal and further relates to the corresponding apparatus. The ionization of dissolved substances by electrospray ionization (ESI) and the ionization of solid substances by matrix-assisted laser desorption (MALDI) have considerably advanced mass spectrometry with respect to the investigation of both small molecules and large biopolymers. In biochemistry, knowledge of the sequence of polymers is particularly important information for the characterization of proteins; in other words, knowledge of the sequence of amino acids. The amino acid sequences form the primary structure of the proteins. Yet the physiological activities and effects of proteins do not depend solely on the sequence. Chains of proteins form so-called secondary structures, such as alpha helices and beta sheets, and these undergo further folding to form tertiary structures, said structures frequently being able to assume several stable configurations. In many cases, these folding structures join together to form complexes and create so-called quaternary structures: hemoglobin, for example, is a characteristic tetramer with two alpha and two beta chains.
Recent investigations into proteins show that neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, ALS (amyotrophic lateral sclerosis), BSE (bovine spongiform encephalopathy) and the related Creutzfeldt-Jakob disease (CJD) are connected with protein misfolding. When investigating the different types of activity of the proteins, it is therefore also very important to obtain information on their folding structures. This information is often equally as important as the information concerning the amino acid sequence. If molecules with the same primary structure possess different folding forms, they are also called “conformation isomers”.
Proteins with different folding structures which are introduced to an organism from outside often produce very different physiological effects. One folding form can have a toxic effect, while another can heal a disease. In the production of pharmaceuticals containing proteins, the quality control used must therefore also include the investigation of the folding structure. It is important to use a method with a large dynamic range of measurement to enable even very small contaminations with toxic substances to be identified.
Geometric changes of the proteins can also be brought about by posttranslational modifications (PTM). Approximately 70 percent of all proteins in the human body are glycosylated. A protein that is modified by polar groups such as phosphates or sugars can undergo structural rearrangements of the main body without losing stability in the process. Posttranslational modifications change the molecular weight of the proteins; phosphorylized or glycosylated proteins have geometric forms which are different to those of their unmodified analogs. Depending on the attachment point of the posttranslational modification, the protein will assume different geometric shapes even though they have the same mass, i.e., the protein will be folded differently. These different types of molecule having the same molecular formula and the same mass but a different primary structure are called “structural isomers”.
Isomers of the primary structure (structural isomers) and isomers of the secondary or tertiary structure (conformation isomers) possess different geometric forms but exactly the same mass. Mass spectrometry is therefore unable to differentiate between them. One of the most efficient methods of identifying and separating such isomers is to separate them by their ion mobility. A cell for the measurement of ion mobility contains an inert gas (such as helium). The ions of the substance under investigation are pulled through the gas by means of an electric field. The high number of collisions with the gas molecules results in a constant drift velocity for each ionic species, which is proportional to the electric field intensity. The proportionality constant is called “ion mobility”. The ion mobility is a function of the temperature, gas pressure, ion charge and, in particular, the collision cross-section. Isomeric ions of the same mass but different collision cross-sections possess different ion mobilities. Isomers with the smallest geometry possess the greatest mobility and therefore the highest drift velocity through the gas. Protein ions in the unfolded state undergo more collisions than tightly folded proteins. Unfolded protein ions therefore arrive at the end of the cell later than folded ions of the same mass.
A variety of information can be obtained from measurements of ion mobility. Simple measurements of relative ion mobility are frequently used to investigate changes in conformation or simply to discover the co-existence of different isomeric structures. Ions with the same mass-to-charge ratio m/z but different folding can be separated from each other relatively easily. It is also possible to determine absolute values for the collision cross-sections (with the extra effort of a calibration). Using the absolute values of the collision cross-sections in a given gas (such as helium) special computer programs can be used to distinguish between different possible folding structures. Such computer programs have become known as AMBER (Assisted Model Building and Energy Refinement) or CHARMM (Chemistry at HARvard Macromolecular Mechanics).
In chemical and biological research, it has become more important to know about the mobility of ions, and therefore devices for the measurement of ion mobility have been incorporated into mass spectrometers in order to combine the measurement of the mass-to-charge ratios of ions with the measurement of collision cross-sections.
The investigation of mass-selected ions with respect to isomeric compounds essentially requires an instrument containing the following components: (1) an ion source, (2) a mass spectrometric ion selector, (3) a measuring cell for the mobility measurement and (4) a time-resolving ion detector.
Often, a separation system for the separation of substances, such as a liquid chromatograph, is additionally used, particularly in conjunction with an electrospray ion source. Selected ions of the same mass are investigated with respect to their mobility; isomers with different mobility then still have the same mass. Therefore, after measuring the mobility, no further mass determination is normally necessary. An ion detector which can measure the ions of different mobilities by temporally resolving the ion current is therefore perfectly satisfactory.
Experiments show, however, that ions can also dissociate in the mobility measuring cell. The mixtures of ions or ion complexes which are of interest for an analysis of the ion mobility often contain quite unstable ions. In this case, not only the intact isomers leave the mobility measuring cell, but also ionized fragmentation products or dissociated ions from complexes, which will normally have different mobilities. Therefore, without a mass spectrometric analysis of the ions after they leave the mobility measuring cell it is not possible to make any statement about the presence of isomers of the mass-selected ions because the occurrence of a number of mobility signals does not permit any conclusion to be drawn about the mass of the ions causing these signals.
U.S. Pat. No. 6,630,662 (A. V. Loboda) discloses an arrangement where the mass filter is located downstream of the mobility measuring cell instead of upstream. The detector then only measures ions with the same mass-to-charge ratio. There could, however, be a fragment ion of a heavier ion with the same mass; this arrangement therefore offers no remedy. Furthermore, this patent specification introduces a moving gas in the mobility measuring cell, which produces a virtual shortening of the measuring cell. Additionally, the ions in the mobility cell are kept on axis by RF-generated pseudopotentials.
This patent also describes an arrangement in which the isomers are subsequently analyzed in a high-resolution time-of-flight mass spectrometer with orthogonal ion injection with the aim of obtaining complete mass spectra of the ion mixtures. Several patents have been elucidated in recent years in relation to this principle of combining the mobility measuring cells with high-resolution time-of-flight mass spectrometers, for example U.S. Pat. No. 5,905,258 (David E. Clemmer and James P. Reilly) and U.S. Pat. No. 6,960,761 (David E. Clemmer). They also describe combinations using time-of-flight mass spectrometers.
Such combinations of mobility measuring cells with orthogonal time-of-flight mass spectrometers (or other kinds of mass spectrometer) raise some problems, however, when the requirement is not just to filter out individual ionic species but to scan whole mass spectra. Time-of-flight mass spectrometers either use ion storage devices before the injection into the time-of-flight mass spectrometer, which destroys the mobility measurement, or they require a continuous ion current, which is not provided by mobility measuring cells. The ion signal of an isomer from an ion mobility measuring cell has a width of only a few hundred microseconds; this is not sufficient time to scan enough mass spectra for a good-quality sum spectrum.
Other types of mass spectrometer, such as ion cyclotron resonance mass spectrometers or RF ion traps, are even less suitable as analyzers for the composition of ions emerging from a mobility measuring cell. The temporal profiles of the signals from the mobility cells are not compatible with the requirements of the mass spectrometers with respect to the temporal duration of the applied ion currents. The only possibility is to collect only particular signals from the mobility cells with a temporal masking and to then subject them to a mass spectrometric analysis. This procedure, however, is not very effective and hardly justifies the development of the complex instrument it requires.
With such instruments, which consist of only a mass filter and an ion mobility measuring cell, the problem remains that the signals from isomeric molecular ions cannot be distinguished from fragment ions or reactively produced ions.